Plasmodium falciparum is a protozoan pathogen that causes the deadliest form of malaria. Malaria has a tremendous impact on human health worldwide, causing nearly one million deaths per year. New therapies are urgently needed to treat this disease, due to widespread chloroquine resistance and emerging resistance to artemisinins. P. falciparum possesses an essential metabolic pathway, non-mevalonate isoprenoid biosynthesis (the MEP pathway), which is not present in humans. This pathway is a particularly enticing antimalarial drug target because it is shared by other important human pathogens, including Gram-negative bacteria and Mycobacterium tuberculosis. The long-term goal is to understand why isoprenoids are essential in malaria parasites. Fosmidomycin is a validated inhibitor of the MEP pathway and is currently in Phase II clinical trials of combination therapy to treat malaria. In preliminary studies, a collection of fosmidomycin-resistant malaria parasites have been developed that not only lack mutations in the known targets of this drug but also continue to grow even when isoprenoid biosynthesis is inhibited. These fosmidomycin-resistant strains presumably survive through genetic changes in a rescue pathway. The objective of this proposal is to determine the biochemical and genetic mechanisms by which these parasites have become resistant. The rationale for these studies is that identification of the genes and pathways that genetically interact with fosmidomycin will inform the regulation and downstream biology of isoprenoid biosynthesis in P. falciparum. Understanding how fosmidomycin-resistant malaria strains survive, despite inhibition of isoprenoid biosynthesis, will elucidate why isoprenoids are typically essential. This approach takes advantage of a pathogen-specific biochemical pathway and a potent chemical inhibitor of isoprenoid biosynthesis that is already in clinical trials. Supported by strong preliminary data that indicate that this strategy wll be successful, the objectives will be met through three specific aims: 1) metabolic analysis of MEP metabolism and protein prenylation (an important function of isoprenoid biosynthesis) in fosmidomycin-resistant malaria parasites; 2) genetic analysis of fosmidomycin-resistant malaria parasites through next-generation sequencing strategies; and 3) identification of the genetic changes that confer fosmidomycin resistance, by recapitulating candidate resistance mutations in sensitive wild-type parasite lines. This approach is innovative, since it uses genetic characterization of drug-resistant malaria parasites not only for drug target validation, but also o expand the fundamental biological understanding of an essential metabolic pathway. The proposed research is significant, because it will identify diagnostic biomarkers of fosmidomycin resistance, improve functional annotation of hypothetical genes in the P. falciparum genome, and identify new targets for much-needed antimalarial drug development.